Experimental investigation of skin tissue-derived extracellular vesicles isolated from type 2 diabetic foot ulcers in impaired wound healing_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Ronghua ¹ , ZHOU Yan ¹ , CHEN Junzhe ¹ , WANG Liangliang ¹ , JIAN Xichao ¹ , QI Fang ^2,3 , DENG Chengliang ^1,2,3 ,  WEI Zairong ^1,2,3,4 ,  LIU Zhiyuan ^1,2,3,4

1. Department of Burns and Plastic Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi Guizhou, 563003, P. R. China;
2. The 2011 Collaborative Innovation Center of Tissue Damage Repair and Regeneration Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi Guizhou, 563003, P. R. China;
3. The Collaborative Innovation Center of Tissue Damage Repair and Regeneration Medicine, Zunyi Medical University, Zunyi Guizhou, 563003, P. R. China;
4. Guizhou Biofabrication Laboratory, Zunyi Guizhou, 563003, P. R. China;

Corresponding?author：

WEI Zairong, Email: zairongwei@163.com; LIU Zhiyuan, Email: zhiyuanliuzx@sina.com

Keywords：

Diabetic foot ulcer; skin tissue; extracellular vesicle; wound repair; macrophage; mice

DOI：

10.7507/1002-1892.202509067

Video：

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Objective To isolate extracellular vesicles (EVs) from the wound-edge skin tissue of type 2 diabetic foot ulcers and to investigate their effect on wound healing in mice. Methods Twenty 8-week-old male db/db mice were used to establish a full-thickness skin defect wound with a diameter of 1.0 cm on the back, and divided into GW4869 (hydrochloride hydrate) group and DMSO group (control group), with 10 mice in each group. The wound healing rate was calculated on days 3, 6, 9, and 12 after injection. HE staining was used to observe epithelialization and inflammatory cell infiltration on days 7 and 12 after injection. Masson staining was used to observe collagen fiber formation. Immunofluorescence staining was used to detect CD206 and interleukin 1β (IL-1β) expressions in wound tissues. Skin tissues from the wound edge of 30 patients with type 2 diabetic foot ulcer were collected for tissue cutting, enzyme dissociation, gradient size exclusion, differential centrifugation combined with ultra-high speed centrifugation to extract tissue EVs (Dia-EVs), which were identified by transmission electron microscopy and particle size analysis. Twenty 8-week-old male C57BL/6 mice were used to establish a full-thickness skin defect wound with a diameter of 1.0 cm on the back, and divided into Dia-EVs group and PBS group, with 10 mice in each group. The wound healing rate was calculated on days 3, 5, 7, 9, and 12 after injection. HE staining was used to observe epithelialization and inflammatory cell infiltration on days 7 and 12 after injection. Masson staining was used to observe collagen fiber formation. Immunofluorescence staining was used to detect CD206 and IL-1β expressions in wound tissues. In vitro experiments were conducted using RAW264.7 cells, which were divided into three groups: the control group [cultured in PRMI 1640 medium containing 10% fetal bovine serum (FBS)], the M1 group [induced with PRMI 1640 medium containing 10%FBS, 200 ng/mL lipopolysaccharide, and 20 ng/mL interferon γ (IFN-γ) for 48 hours], and the Dia-EVs group (treated with PRMI 1640 medium containing 10%FBS and 40 μg/mL Dia-EVs for 48 hours). The expression levels of CD206 and IL-1β in each group of cells were observed. Results In db/db mice experiment, on days 3, 6, 9, and 12 after injection, the wound healing rate of the GW4869 group was significantly faster than that of the DMSO group (P<0.05). On day 7 after injection, inflammatory cell infiltration in the DMSO group was obvious, and the number of inflammatory cells was significantly higher than that in the GW4869 group (P<0.05). Masson staining showed no obvious new collagen formation in either group. On day 12 after injection, HE staining showed that the GW4869 injection group had completed epithelialization without obvious inflammatory cell infiltration, while the DMSO injection group still had a large number of inflammatory cells infiltrating (P<0.05). Moreover, a large amount of new collagen was formed in the GW4869 group, and the collagen volume ratio was significantly greater than that of the DMSO group (P<0.05). On 7 days after injection, no significant CD206 positive expression was observed in either group, but a large amount of IL-1β positive expression was seen. Moreover, the IL-1β relative expression in the DMSO group was significantly more than that in the GW4869 group (P<0.05). On 12 days after injection, CD206 positive expression was observed in the GW4869 group, while no significant expression was seen in the DMSO group (P<0.05); no obvious IL-1β positive expression was found in the GW4869 group, while the DMSO group still had a large amount of IL-1β positive expression (P<0.05). In C57BL/6 mice experiment, on days 3, 5, 7, 9, and 12 after injection, the wound healing rate of the Dia-EVs group was significantly slower than that of the PBS group (P<0.05). On day 7 after injection, inflammatory cell infiltration in the Dia-EVs group was obvious, and the number of inflammatory cells was significantly higher than that in the PBS group (P<0.05). Masson staining showed no obvious new collagen formation in either group. On day 12 after injection, wound defects still existed in both the Dia-EVs group and the PBS group, but the wound area in the PBS group was narrower. The number of inflammatory cells showed no significant difference (P>0.05), while a large amount of new collagen formation was observed in the PBS group, significantly more than that in the Dia-EVs group (P<0.05). Immunofluorescence results showed that on days 7 and 12 after injection, the number of IL-1β-positive cells in the Dia-EVs group was significantly higher than that in the PBS group (P<0.05), while no significant CD206 positive expression was observed in either group (P>0.05). In vitro experimental results showed that there was no significant difference in the relative expression of CD206 among the three groups (P>0.05). The relative expressions of IL-1β in M1 group and Dia-EVs group were significantly higher than that in the control group (P<0.05), but there was no significant difference between the two groups (P>0.05).Conclusion Dia-EVs delay wound healing in mice by inducing M1 polarization of macrophages, inhibiting M2 transformation, and reducing collagen deposition.

Citation： LI Ronghua, ZHOU Yan, CHEN Junzhe, WANG Liangliang, JIAN Xichao, QI Fang, DENG Chengliang, WEI Zairong, LIU Zhiyuan. Experimental investigation of skin tissue-derived extracellular vesicles isolated from type 2 diabetic foot ulcers in impaired wound healing. Chinese Journal of Reparative and Reconstructive Surgery, 2026, 40(4): 664-674. doi: 10.7507/1002-1892.202509067 Copy

1.	Xiong Y, Chen L, Yan C, et al. Circulating exosomal miR-20b-5p inhibition restores Wnt9b signaling and reverses diabetes-associated impaired wound healing. Small, 2020, 16(3): e1904044. doi: 10.1002/smll.201904044.
2.	喻先軍, 張定偉, 余琳, 等. 脛骨橫向骨搬移術治療Wagner 3~4級2型糖尿病足潰瘍的臨床療效及免疫球蛋白水平變化研究. 中國修復重建外科雜志, 2025, 39(8): 1030-1036.
3.	Li D, Wu N. Mechanism and application of exosomes in the wound healing process in diabetes mellitus. Diabetes Res Clin Pract, 2022, 187: 109882. doi: 10.1016/j.diabres.2022.109882.
4.	Chen Y, Yin W, Liu Z, et al. Exosomes derived from fibroblasts enhance skin wound angiogenesis by regulating HIF-1α/VEGF/VEGFR pathway. Burns Trauma, 2025, 13: tkae071. doi: 10.1093/burnst/tkae071.
5.	Kuang L, Zhang C, Li B, et al. Human keratinocyte-derived exosomal MALAT1 promotes diabetic wound healing by upregulating MFGE8 via microRNA-1914-3p. Int J Nanomedicine, 2023, 8: 949-970.
6.	Yi J, Tang Q, Sun S, et al. Exosomes in diabetic wound healing: Mechanisms, applications, and perspectives. Diabetes Metab Syndr Obes, 2025, 18: 2955-2976.
7.	王江文, 易陽艷, 朱元正, 等. 脂肪干細胞來源外泌體促進糖尿病小鼠創面愈合的實驗研究. 中國修復重建外科雜志, 2020, 34(1): 124-131.
8.	Song J, Zhao T, Wang C, et al. Cell migration in diabetic wound healing: Molecular mechanisms and therapeutic strategies (Review). Int J Mol Med, 2025, 56(2): 126. doi: 10.3892/ijmm.2025.5567.
9.	Estrada AL, Valenti ZJ, Hehn G, et al. Extracellular vesicle secretion is tissue-dependent ex vivo and skeletal muscle myofiber extracellular vesicles reach the circulation in vivo. Am J Physiol Cell Physiol, 2022, 322(2): C246-C259.
10.	Guda PR, Sharma A, Anthony AJ, et al. Nanoscopic and functional characterization of keratinocyte-originating exosomes in the wound fluid of non-diabetic and diabetic chronic wound patients. Nano Today, 2023, 52: 101954. doi: 10.1016/j.nantod.2023.101954.
11.	Lee JC, Ray RM, Scott TA. Prospects and challenges of tissue-derived extracellular vesicles. Mol Ther, 2024, 32(9): 2950-2978.
12.	王珍. 模擬創面微環境誘導的人臍帶間充質干細胞外泌體對創面愈合的影響研究. 遵義: 遵義醫科大學, 2022.
13.	Welsh JA, Goberdhan DCI, O’Driscoll L, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles, 2024, 13(2): e12404. doi: 10.1002/jev2.12404.
14.	Saadh MJ, Allela OQB, Kareem RA, et al. Harnessing exosomal mediators for advanced wound healing: Mechanisms and therapeutic potential in angiogenesis. Microvasc Res, 2025, 162: 104861. doi: 10.1016/j.mvr.2025.104861.
15.	Jo C, Choi YJ, Lee TJ. Therapeutic potential of stem cell-derived exosomes in skin wound healing. Biomimetics (Basel), 2025, 10(8): 546. doi: 10.3390/biomimetics10080546.
16.	Crescitelli R, L?sser C, L?tvall J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat Protoc, 2021, 16(3): 1548-1580.
17.	Jin W, Li Y, Yu M, et al. Advances of exosomes in diabetic wound healing. Burns Trauma, 2025, 13: tkae078. doi: 10.1093/burnst/tkae078.
18.	Lin J, Lu W, Huang B, et al. The role of tissue-derived extracellular vesicles in tumor microenvironment. Tissue Cell, 2024, 89: 102470. doi: 10.1016/j.tice.2024.102470.
19.	Wang J, Li L, Zhang Z, et al. Extracellular vesicles mediate the communication of adipose tissue with brain and promote cognitive impairment associated with insulin resistance. Cell Metab, 2022, 34(9): 1264-1279.
20.	Jiang F, Chen Q, Wang W, et al. Hepatocyte-derived extracellular vesicles promote endothelial inflammation and atherogenesis via microRNA-1. J Hepatol, 2020, 72(1): 156-166.
21.	Shaihov-Teper O, Ram E, Ballan N, et al. Extracellular vesicles from epicardial fat facilitate atrial fibrillation. Circulation, 2021, 143(25): 2475-2493.
22.	Zheng S, Zeng Y, Chu L, et al. Renal tissue-derived exosomal miRNA-34a in diabetic nephropathy induces renal tubular cell fibrosis by promoting the polarization of M1 macrophages. IET Nanobiotechnol, 2024, 2024: 5702517. doi: 10.1049/2024/5702517.
23.	Deng W, Zhu X, Li H, et al. Lung tissue extracellular vesicles-mediated delivery of miR-128-3p as a novel mechanism of acute lung inflammation. Int J Nanomedicine, 2025, 20: 4831-4848.
24.	Gao F, He Y, Xue Q, et al. Fine particulate matter contributes to the development of atherosclerosis via miR-3529-3p encapsulated in extracellular vesicles. J Hazard Mater, 2025, 496: 139508. doi: 10.1016/j.jhazmat.2025.139508.
25.	Jeong I, Lee J, Park SJ, et al. High fat diet enhances catalase loading into adipose tissue derived extracellular vesicles with limited effect on oxidative stress. Sci Rep, 2025, 15(1): 31010. doi: 10.1038/s41598-025-15594-5.
26.	Huang Y, Abdelgawad A, Turchinovich A, et al. RNA landscapes of brain and brain-derived extracellular vesicles in simian immunodeficiency virus infection and central nervous system pathology. J Infect Dis, 2024, 229(5): 1295-1305.
27.	Li H, Liu Y, Lin Y, et al. Cardiac repair using regenerating neonatal heart tissue-derived extracellular vesicles. Nat Commun, 2025, 16(1): 1292. doi: 10.1038/s41467-025-56384-x.
28.	Lee J, Kim SR, Lee C, et al. Extracellular vesicles from in vivo liver tissue accelerate recovery of liver necrosis induced by carbon tetrachloride. J Extracell Vesicles, 2021, 10(10): e12133. doi: 10.1002/jev2.12133.
29.	Yoon YJ, Bae S, Choi EJ, et al. Mouse tumor tissue-derived extracellular vesicles induce angiogenesis through VEGF production from macrophages. J Extracell Vesicles, 2025, 14(8): e70138. doi: 10.1002/jev2.70138.
30.	Lou P, Liu S, Wang Y, et al. Neonatal-tissue-derived extracellular vesicle therapy (NEXT): A potent strategy for precision regenerative medicine. Adv Mater, 2023, 35(33): e2300602. doi: 10.1002/adma.202300602.
31.	Jiang P, Ma X, Wang X, et al. Isolation and comprehensive analysis of cochlear tissue-derived small extracellular vesicles. Adv Sci (Weinh), 2024, 11(48): e2408964. doi: 10.1002/advs.202408964.
32.	No authors listed. Correction to “Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches”. J Extracell Vesicles, 2024, 13(5): e12451. doi: 10.1002/jev2.12451.
33.	Wang F, Yao J, Zuo H, et al. Diverse-origin exosomes therapeutic strategies for diabetic wound healing. Int J Nanomedicine, 2025, 20: 7375-7402.
34.	Cheng Y, Wang Y, Wang Y, et al. Microenvironment-feedback regulated hydrogels as living wound healing materials. Nat Commun, 2025, 16(1): 6050. doi: 10.1038/s41467-025-60858-3.
35.	Cao J, Zhang X, Li Z, et al. Adipose-derived mesenchymal stem cells accelerate diabetic foot ulcer healing by promoting macrophage M2 polarization through downregulation of EREG and CSTA. J Inflamm Res, 2025, 18: 7749-7768.
36.	Boodhoo K, Vlok M, van de Vyver M. A macrophage-based cell therapy approach promotes collagen deposition in diabetic wounds. Wound Repair Regen, 2025, 33(4): e70071. doi: 10.1111/wrr.70071.

1. Xiong Y, Chen L, Yan C, et al. Circulating exosomal miR-20b-5p inhibition restores Wnt9b signaling and reverses diabetes-associated impaired wound healing. Small, 2020, 16(3): e1904044. doi: 10.1002/smll.201904044.
2. 喻先軍, 張定偉, 余琳, 等. 脛骨橫向骨搬移術治療Wagner 3~4級2型糖尿病足潰瘍的臨床療效及免疫球蛋白水平變化研究. 中國修復重建外科雜志, 2025, 39(8): 1030-1036.
3. Li D, Wu N. Mechanism and application of exosomes in the wound healing process in diabetes mellitus. Diabetes Res Clin Pract, 2022, 187: 109882. doi: 10.1016/j.diabres.2022.109882.
4. Chen Y, Yin W, Liu Z, et al. Exosomes derived from fibroblasts enhance skin wound angiogenesis by regulating HIF-1α/VEGF/VEGFR pathway. Burns Trauma, 2025, 13: tkae071. doi: 10.1093/burnst/tkae071.
5. Kuang L, Zhang C, Li B, et al. Human keratinocyte-derived exosomal MALAT1 promotes diabetic wound healing by upregulating MFGE8 via microRNA-1914-3p. Int J Nanomedicine, 2023, 8: 949-970.
6. Yi J, Tang Q, Sun S, et al. Exosomes in diabetic wound healing: Mechanisms, applications, and perspectives. Diabetes Metab Syndr Obes, 2025, 18: 2955-2976.
7. 王江文, 易陽艷, 朱元正, 等. 脂肪干細胞來源外泌體促進糖尿病小鼠創面愈合的實驗研究. 中國修復重建外科雜志, 2020, 34(1): 124-131.
8. Song J, Zhao T, Wang C, et al. Cell migration in diabetic wound healing: Molecular mechanisms and therapeutic strategies (Review). Int J Mol Med, 2025, 56(2): 126. doi: 10.3892/ijmm.2025.5567.
9. Estrada AL, Valenti ZJ, Hehn G, et al. Extracellular vesicle secretion is tissue-dependent ex vivo and skeletal muscle myofiber extracellular vesicles reach the circulation in vivo. Am J Physiol Cell Physiol, 2022, 322(2): C246-C259.
10. Guda PR, Sharma A, Anthony AJ, et al. Nanoscopic and functional characterization of keratinocyte-originating exosomes in the wound fluid of non-diabetic and diabetic chronic wound patients. Nano Today, 2023, 52: 101954. doi: 10.1016/j.nantod.2023.101954.
11. Lee JC, Ray RM, Scott TA. Prospects and challenges of tissue-derived extracellular vesicles. Mol Ther, 2024, 32(9): 2950-2978.
12. 王珍. 模擬創面微環境誘導的人臍帶間充質干細胞外泌體對創面愈合的影響研究. 遵義: 遵義醫科大學, 2022.
13. Welsh JA, Goberdhan DCI, O’Driscoll L, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles, 2024, 13(2): e12404. doi: 10.1002/jev2.12404.
14. Saadh MJ, Allela OQB, Kareem RA, et al. Harnessing exosomal mediators for advanced wound healing: Mechanisms and therapeutic potential in angiogenesis. Microvasc Res, 2025, 162: 104861. doi: 10.1016/j.mvr.2025.104861.
15. Jo C, Choi YJ, Lee TJ. Therapeutic potential of stem cell-derived exosomes in skin wound healing. Biomimetics (Basel), 2025, 10(8): 546. doi: 10.3390/biomimetics10080546.
16. Crescitelli R, L?sser C, L?tvall J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat Protoc, 2021, 16(3): 1548-1580.
17. Jin W, Li Y, Yu M, et al. Advances of exosomes in diabetic wound healing. Burns Trauma, 2025, 13: tkae078. doi: 10.1093/burnst/tkae078.
18. Lin J, Lu W, Huang B, et al. The role of tissue-derived extracellular vesicles in tumor microenvironment. Tissue Cell, 2024, 89: 102470. doi: 10.1016/j.tice.2024.102470.
19. Wang J, Li L, Zhang Z, et al. Extracellular vesicles mediate the communication of adipose tissue with brain and promote cognitive impairment associated with insulin resistance. Cell Metab, 2022, 34(9): 1264-1279.
20. Jiang F, Chen Q, Wang W, et al. Hepatocyte-derived extracellular vesicles promote endothelial inflammation and atherogenesis via microRNA-1. J Hepatol, 2020, 72(1): 156-166.
21. Shaihov-Teper O, Ram E, Ballan N, et al. Extracellular vesicles from epicardial fat facilitate atrial fibrillation. Circulation, 2021, 143(25): 2475-2493.
22. Zheng S, Zeng Y, Chu L, et al. Renal tissue-derived exosomal miRNA-34a in diabetic nephropathy induces renal tubular cell fibrosis by promoting the polarization of M1 macrophages. IET Nanobiotechnol, 2024, 2024: 5702517. doi: 10.1049/2024/5702517.
23. Deng W, Zhu X, Li H, et al. Lung tissue extracellular vesicles-mediated delivery of miR-128-3p as a novel mechanism of acute lung inflammation. Int J Nanomedicine, 2025, 20: 4831-4848.
24. Gao F, He Y, Xue Q, et al. Fine particulate matter contributes to the development of atherosclerosis via miR-3529-3p encapsulated in extracellular vesicles. J Hazard Mater, 2025, 496: 139508. doi: 10.1016/j.jhazmat.2025.139508.
25. Jeong I, Lee J, Park SJ, et al. High fat diet enhances catalase loading into adipose tissue derived extracellular vesicles with limited effect on oxidative stress. Sci Rep, 2025, 15(1): 31010. doi: 10.1038/s41598-025-15594-5.
26. Huang Y, Abdelgawad A, Turchinovich A, et al. RNA landscapes of brain and brain-derived extracellular vesicles in simian immunodeficiency virus infection and central nervous system pathology. J Infect Dis, 2024, 229(5): 1295-1305.
27. Li H, Liu Y, Lin Y, et al. Cardiac repair using regenerating neonatal heart tissue-derived extracellular vesicles. Nat Commun, 2025, 16(1): 1292. doi: 10.1038/s41467-025-56384-x.
28. Lee J, Kim SR, Lee C, et al. Extracellular vesicles from in vivo liver tissue accelerate recovery of liver necrosis induced by carbon tetrachloride. J Extracell Vesicles, 2021, 10(10): e12133. doi: 10.1002/jev2.12133.
29. Yoon YJ, Bae S, Choi EJ, et al. Mouse tumor tissue-derived extracellular vesicles induce angiogenesis through VEGF production from macrophages. J Extracell Vesicles, 2025, 14(8): e70138. doi: 10.1002/jev2.70138.
30. Lou P, Liu S, Wang Y, et al. Neonatal-tissue-derived extracellular vesicle therapy (NEXT): A potent strategy for precision regenerative medicine. Adv Mater, 2023, 35(33): e2300602. doi: 10.1002/adma.202300602.
31. Jiang P, Ma X, Wang X, et al. Isolation and comprehensive analysis of cochlear tissue-derived small extracellular vesicles. Adv Sci (Weinh), 2024, 11(48): e2408964. doi: 10.1002/advs.202408964.
32. No authors listed. Correction to “Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches”. J Extracell Vesicles, 2024, 13(5): e12451. doi: 10.1002/jev2.12451.
33. Wang F, Yao J, Zuo H, et al. Diverse-origin exosomes therapeutic strategies for diabetic wound healing. Int J Nanomedicine, 2025, 20: 7375-7402.
34. Cheng Y, Wang Y, Wang Y, et al. Microenvironment-feedback regulated hydrogels as living wound healing materials. Nat Commun, 2025, 16(1): 6050. doi: 10.1038/s41467-025-60858-3.
35. Cao J, Zhang X, Li Z, et al. Adipose-derived mesenchymal stem cells accelerate diabetic foot ulcer healing by promoting macrophage M2 polarization through downregulation of EREG and CSTA. J Inflamm Res, 2025, 18: 7749-7768.
36. Boodhoo K, Vlok M, van de Vyver M. A macrophage-based cell therapy approach promotes collagen deposition in diabetic wounds. Wound Repair Regen, 2025, 33(4): e70071. doi: 10.1111/wrr.70071.

Chinese Journal of Reparative and Reconstructive Surgery

Experimental investigation of skin tissue-derived extracellular vesicles isolated from type 2 diabetic foot ulcers in impaired wound healing

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